The Canine Parvovirus (CPV) belongs to the autonomous parvoviruses, causing severe enteritis in dogs of all ages and myocarditis in puppies less than 12 weeks old. CPV was first isolated in 1978 (Burtonboy, G. et al., Arch. Virol. 61:1-11, 1979; Appel et al., Vet. Rec. 105. 156-179, 1979). It is believed to have arisen as a natural variant of the feline panleukopenia virus (FPLV) or the mink enteritis virus (MEV). CPV infection is controlled using conventional vaccines based on live or inactivated viruses. However, since dogs are vaccinated before pregnancy, maternal antibodies can block attenuated live vaccine replication. Autonomous parvoviruses provide a good vector for recombinant subunit vaccine production for several reasons, inter alia:
1. The capsid proteins are structurally simple (no glycosylation, phosphorylation or acetylation is required). PA0 2. Humoral response appears to adequately control viral dissemination given the relative efficiency of inactivated vaccines. PA0 1. LucKow, V. A. & Summers, M. D. (1988). Trends in the development of baculovirus expression vectors. Bio/Technology 6, 47-55. PA0 2. J. Vialard et al. (1990). Synthesis of the membrane fusion and Hemagglutinin Proteins of Measles virus, using a novel baculovirus vector containing the .beta.-galactosidase gene. J. Virol. 64. 37-50. PA0 i) the CPV VP2 protein produced in an expression vector of baculoviruses multiplied in a cell culture of a permissive host (this protein hereinafter optionally referred to as "VP2 hereof"); or PA0 ii) empty VP2 capsids formed by assembly of the VP2 hereof.
Protein and DNA sequence studies and serologic studies show an enhanced antigenic and genetic homology between CPV, FPLV, MEV and the Raccoon Parvovirus (Tratschin et al., J. Gen Virol. 61:33-41. 1982. Carlsson et al., J. Virol., 55, 574-582, 1985. Parrish et al., Arch. Virol. 72, 267-278, 1982. Reed et al., J. Virol. 62:266-276, 1988). Despite this homology they are exquisitely specific in the "in vivo" host, although "in vitro" all viruses replicate in cat kidney cells (Appel et al., Vet. Rec. 105, 156-179, 1979. Trastschin et al., J. Gen. Virol., 61:33-41, 1982). The CPV capsid contains two proteins with broadly overlapping amino acid sequences, VP1 (82-84 KDa) and VP2 (67.70 KDa) (Paradiso et al. J. Gen. Virol. 39, 800-807, 1982. Surleraux et al. Arch Virol., 82, 233-240, 1984. J. Gen. Virol. 62, 113-125, 1982. Surleraux et al., Arch. Virol. 82, 233-240, 1984). The parvovirus capsid has a diameter of 22 nm and holds some 10 VP1 copies and some 60 VP2 copies (Wobble et al., Biochemistry 23, 6565-6569, 1984), arranged as either homo- or heterodimers (Paradiso, J. Virol., 46, 94-102, 1983) though the precise capsid structure is unknown. VP2 in full capsids (holding DNA) is preferentially broken down by proteolytic digestion into 63-67 KDa VP3 (Paradiso et al., Gen. Virol. 39, 800-807, 1982. Surleaux et al. Arch. Virol., 82, 233.240, 1984) after capsid assembly (Paradiso, J. Virol., 39, 800-807, 1981).
Our laboratory has of late been researching into the immunogenicity of various fragments of the proteins making up the CPV viral capsid which has resulted in new VP2 protein and VP2 and VP1 fragment-based recombinant vaccines being described. These findings are summarised in Spanish patent application number 9002074 for: "PRODUCTION OF CANINE PARVOVIRUS VACCINES BY VIRAL POLYPEPTIDE EXPRESSION IN E. coli OR BY CHEMICAL SYNTHESIS" with J. I. Casal et al. as inventors.
The said patent application relates to the expression of such products in E. coli bacterial vectors.
New large-scale protein production vectors have however been recently discovered based upon the replication of recombinant baculoviruses derived from the Autographa californica nuclear polyhedrosis virus (ACMNPV) in culture insect cells. The state of the art for these vectors is summed up in two scientific papers as follows:
Furthermore, European Patent Application number 0 341 611 by Cornell Research Foundation, Inc. and Boyce Thompson Institute for Plant Research, Inc., describes the obtention of CPV subunit vaccines using a baculovirus expression vector other than that used in the present invention, and the said European Patent moreover fails to mention that the proteins obtained are capable of forming empty VP2 capsids thereby drawing a fundamental distinction with the instant invention inasmuch as the VP2 protein obtained under our invention is able to form empty VP2 capsids. In consequence of the above their immunogenic and hemagglutinant capacity is clearly superior as the following description will show. This added capacity of the VP2 protein obtained with our invention is further convenient in that other viral protein epitopes can be introduced in the said capsids by genetic manipulation of the recombinant baculoviruses, or by chemical manipulation of the actual capsids.
The advantages of VP2 protein synthesis in a baculovirus vector over E. coli synthesis are remarkable, namely a greater fidelity as to the identity of the protein obtained, a greater solubility of the product obtained, fusion proteins need not be used, etc. These are clearly advancements of and improvement to processes for obtaining recombinant subunit vaccines as regards previous processes.